Abstract Over the past decade, RNA-mediated regulation has emerged as a central theme in gene expression. A common control mechanism of Gram-positive bacteria is the riboswitch. In riboswitches, a sensor domain is mechanistically coupled to a regulatory domain composed of mutually exclusive terminator/anti-terminator RNA structure elements that either attenuate or increase translation/transcription. A particularly widespread type of riboswitch is the T box, a tRNA-actuated switch whose default state is to attenuate transcription of essential genes encoding aminoacyl-tRNA synthetases, amino acid biosynthetic enzymes, and amino acid transport machinery. High ratios of charged-to-uncharged tRNA maintain the switch in the off state and suppress gene expression. The performance of the riboswitch is subject to modulation by multiple factors, including nucleotide sequence variations around conserved structure elements and tRNA base modification, and is crucial to the fitness of the cell. In this proposal, we will (1) identify the global conformationsand structural details of model tRNA-riboswitch complexes using SAXS and solution NMR methods; (2) develop a kinetic model of the T box riboswitch mechanism that includes variables such as tRNA modification and aminoacylation state using single molecule FRET techniques; and (3) determine in vivo the effects of tRNA modification and natural sequence variation on T box regulatory efficiency using direct RNA measurement assays. For the first time perhaps for any riboswitch, we will establish a mechanistic framework for riboswitch function that quantitatively connects local sequence-dependent contributions with global dynamics and T box efficiency. In addition, this project will broaden our knowledge of the contributions of tRNA nucleotide modification to gene regulation and cell fitness of Gram-positive bacteria. Finally, the T box riboswitch offers a unique platform on which to engineer genetic tools to interrogate the metabolic state of bacterial cells.